Hospital-acquired pathogenic microbial infections require rapid identification of the microbe(s) present to ensure appropriate antimicrobial treatment. On any given day, about 1 in 31 hospital patients has at least one hospital-acquired pathogenic microbial infection. In 2015, there were an estimated 687,000 hospital-acquired pathogenic microbial infections, resulting in 72,000 deaths. Administration of the broad-spectrum, non-targeted antimicrobials to patients with these hospital-acquired microbial infections can lead to antimicrobial resistance.
The preservation of microbes in clinical samples obtained from subjects with pathogenic microbial infections is critical for subsequent clinical diagnostic tests. To that end, polyanionic detergents are effective for inhibiting bactericidal processes in whole blood, such as the complement pathway, and are commonly used in blood culture bottles to promote the survival and growth of bacteria in samples (Edberg et al., 1976, “Use of sodium polyanethol sulfonate to selectively inhibit aminoglycoside and polymyxin antibiotics in a rapid blood level antibiotic assay,” Antimicro. Agents Chemother. 9(3): 414-417; Kocka et al., 1972, “Action of sulfated polyanions used in blood culture on lysozyme, complement, and antibiotics,” Ann. Clinc. Lab. Sci. 2(6): 470-473; Traub and Kleber, 1977, “Inactivation of classical and alternative pathway-activated bactericidal activity of human serum by sodium polyanethol sulfonate,” J. Clin. Microbiol. 5(3): 278-284).
Next-generation molecular diagnostics (e.g., nucleic acid amplification) for the identification of bacteria from clinical blood samples offers a rapid alternative to the traditional cultivation of bacteria. However, polyanionic compounds which are commonly added to clinical samples, such as sodium polyanethol sulfonate (SPS) to inhibit bactericidal processes and heparin to inhibit clotting, have an inhibitory effect on nucleic acid amplification techniques (Fredericks and Relman, 1998, “Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanethol sulfonate,” J. Clin. Microbiol., 36(10): 2810-2816; Qian et al., 2001, “Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results,” J. Clin. Microbiol., 39(10: 3578-3585; and Regan et al., 2012, “A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture,” J. Mol. Diagn. 14(2): 120-129). In addition to such additives, blood components such as hemoglobin, lactoferrin, heme, and immunoglobulins can also interfere with nucleic acid amplification procedures. Chemical similarities between nucleic acids and polyanionic polymers such as SPS, however, lead to the co-purification of both polymers in multiple nucleic acid purification techniques, thereby resulting in inhibition of downstream molecular applications (Regan et al., 2012, supra).
Improved methods are needed to reduce the negative effects of certain common additives and naturally-occurring compounds (e.g., SPS, heparin) on nucleic acid amplification-based diagnostics during the processing of clinical samples. Current methods of clinical sample processing for downstream nucleic acid amplification currently dilute final samples from 10× to 1000× to mitigate SPS inhibition. However, these approaches also significantly reduce nucleic acid detection sensitivity (Pennington, 2014, “Dealing with amplification inhibitors: reagent choice matters,” Promega Corporation).